Conifer trees (Pinophyta, or softwoods) are cone-bearing gymnosperm species. Conifer species are particularly characterized by production of resin containing a high concentration of terpenes, especially as compared to hardwood species. This resin is considered one of the oldest and most important non-wood products derived from conifer trees, which is used to produce a number of products including turpentine and rosin. Many of the monoterpenoids that make up the terpene fraction of conifer resin are known to be highly inhibitory to the growth of wood decomposing fungi, including alpha-pinene, beta-pinene, limonene, camphene, and myrcen]. The inhibitory activity of conifer terpenes on wood decomposing fungi functions as a natural defense against fungal infection. As a result, wood decomposing fungi are typically characterized by their occurrence on either hardwood and/or conifer wood species, governed in part by varying levels of tolerance to conifer terpenes.
Solid-state cultivation of fungi on organic matter typically requires heat sterilization/pasteurization in order to eliminate/reduce ambient bioburden (yeast, mold, bacteria) present in the given substrate. This bioburden, if not eliminated prior to inoculation with the fungus species, will compete with the fungus for nutrition leading to partial or total inhibition of mycelium production. Additionally, most fungus cultivation procedures require that incubation occur in an aseptic arena to prevent accidental introduction of contaminant species post-sterilization/pasteurization (i.e. aseptic cultivation).
In the mushroom cultivation industry, the use of conifer substrates is largely avoided. This is due to two factors, the first being terpene inhibition as described above. Secondly, conifer wood provides poor nutrition for supporting rapid fungal growth, with specific deficiencies in nitrogen and trace minerals. Commercial cultivation of mushrooms on conifer substrates is rare, and often utilizes tree species with low resin content (such as Douglas Fir) blended 1:1 with non-conifer substrates (hardwood, agricultural waste) and high-value supplemental nutrition (wheat bran, brown rice flour) within cultivation systems that require aseptic cultivation as described above. Though in practice the use of conifer substrates for mushroom cultivation is rare, there are a multitude of patents, including U.S. Pat. No. 7,984,584, indicating the use of conifer substrates for mushroom cultivation, particularly for shimenji mushroom production. In this case, typical supplementation rates with non-conifer species and high value nutrition are 50 to >60%.
Accordingly, it is an object of the invention to provide an economically feasible process for solid-state cultivation of mycelium on a lignocellulose substrate.
It is another object of the invention to reduce the cost of processing mycelium on a lignocellulose substrate.
It is another object of the invention to provide a mycelium-based biomaterial for use in the engineered wood industry.
Briefly, the invention provides a solid-state process wherein mycelium is cultivated on a lignocellulose substrate and, particularly, a conifer/gymnospermous substrate.
The process leverages the inhibitory nature of conifer and terpene containing substrates, in concert with specific fungal strain selection criteria and sequence of mycelium expansion and nutrition supplementation, to reduce the need for aseptic cultivation procedures to a maximum of 15% of the total mass of substrate being myceliated.
The process proposes a maximum supplemental nutrition rate of 14%, which significantly reduces cost as compared to other patents in the field of mushroom cultivation. Further, the methodology employed leverages a very specific sequence of staged mycelium expansion and rationed nutritional supplementation that selects for a desired fungal species over that of contaminant organisms (mold, bacteria, yeast). Within this process, the total mass requiring heat-processing and aseptic control is reduced to a maximum of 15% which provides for unique processing efficiency as compared to the current state of the art of mushroom cultivation.
In the context of manufacturing a biomaterial by growing mycelium on a solid substrate, a primary cost driver is processing time (growth time), which is governed by the rate at which the fungus expands (grows) through the solid substrate. When presented with the problem of growing mycelium on a conifer substrate, the most obvious assumption would be to utilize fungus species that occur exclusively on conifer trees in nature. Research found the opposite to be true; in trials conducted with a multitude of fungal and conifer wood species, it was found that fungal species which primarily colonize hardwoods in nature, and are tolerant of the terpene-containing fraction of the conifer substrate, had expansion rates (the rate at which the mycelium grows through the given substrate) 55%-61% faster than species that occur on conifers exclusively. As a result, this study indicated that the most attractive species in regard to processing time are those that meet the criteria described in process step 1 below. Additionally, the increase in the rate of expansion associated with the selection criteria of 1 was found to be critical for successful exclusion of contaminant organisms; therefore the specific combination of fungal strain selection, substrate species selection, and the specific sequence in which the fungus is expanded in concert with introduction of supplemental nutrition described in the proposed process is critical for achieving both the desired processing efficiency and material performance.
There are several patented and common processes oriented toward growing mycelium on non-sterilized wood substrate without maintaining aseptic control (i.e. non-aseptic cultivation). These processes differ in important and significant ways from the proposed process in both methodology, result, and application as detailed below:                1. Log mushroom cultivation: It is common mushroom cultivation practice to inoculate intact hardwood logs for the purposes of outdoor mushroom cultivation. This process is dependent on using intact logs, specifically leveraging the bark along with added wax to exclude contaminant organisms. Therefore, the process is not applicable to particulate substrates. Additionally, this process specifically teaches not to use logs from softwoods in most scenarios, requires processing times often exceeding 1 year, and does not produce adequate biomass for material applications.        2. Biopulping: This process teaches the utilization of white rot fungi to degrade lignin in wood chips prior to mechanical pulping in the paper manufacturing industry, thereby reducing the energy required to effectively pulp the chips. The biopulping process does not require aseptic incubation conditions, but is also not specifically concerned with exclusion of other fungi/mold species as the proposed process is. Additionally, this process requires heat disinfection of the entire mass of substrate being processed, whereas the proposed process only heat disinfects a maximum of 15% of the wood particles processed. Furthermore, the specific fungal strains utilized in biopulping as demonstrated in testing performed at Ecovative do not perform adequately as a bioresin, whereas the strain selection criteria detailed here selects for species which do perform adequately as a bioresin.        3. Cartapip97]: The product Cartapip97 is intended for inoculating particulate conifer and hardwood substrates without sterilization or aseptic control. Similar to biopulping this product is designed to effect a specific effect on the wood substrate rather than accumulate fungal biomass; the only application is to reduce the effect of sapstain fungi on wood particles and logs. The proposed methodology specifically teaches the negotiation of supplemental nutrition relative to fungal expansion to (a) exclude contaminant organisms, (b) accumulate at least 10% fungal biomass, and (c) utilize fungal species with morphological characteristics that provide value as a bioresin. Cartapip97 does not leverage supplemental nutrition for biomass accumulation and only teaches to a specific effect on substrate with a specific fungal species, not to the functionality of the biomass itself as a material.        
In accordance with the process of the invention, after obtaining a lignocellulose substrate, a fraction of the substrate of up to 15% is combined with supplemental nutritional material at a ratio of up to 14% of the dry mass of the fraction and hydrated to a moisture content of from 40% to 70% by weight.
The hydrated substrate fraction is then heat processed for a period of time sufficient to remove ambient bioburden (yeast, mold, bacteria) and to maintain the hydrated substrate fraction in an aseptic condition.
Thereafter, the hydrated substrate fraction is inoculated with a fungus and incubated for a period of time to allow the fungus to grow hyphae and to allow the hyphae to form a network of interconnected mycelia cells through and around discrete particles of the substrate fraction to obtain a myceliated substrate.
Next, the myceliated substrate is reduced into discrete particles, for example, by agitation, grinding or otherwise.
The remaining fraction of the substrate is then combined with water to obtain a moisture content of 40% to 70%; then combined with the discrete particles of myceliated substrate; and incubated for a period of time to allow the fungus in the discrete particles of the myceliated substrate to grow hyphae and to allow the hyphae to form a network of interconnected mycelia cells through and around discrete particles of the remaining fraction of the substrate to obtain a second myceliated substrate.
The second myceliated substrate is then reduced into discrete particles; combined with supplemental nutritional material at a ratio of up to 14% of the dry mass of the second myceliated substrate; and incubated for a period of time to allow the fungus in the discrete particles of myceliated substrate to grow hyphae and to allow the hyphae to form a network of interconnected mycelia cells through and around discrete particles of the remaining fraction of the substrate to obtain a third myceliated substrate composed of at least 10% mycelium.